Isolation and characterization of a N. crassa silencing gene and uses thereof

ABSTRACT

A nucleotide sequence encoding for a protein characterized in that it has a silencing activity and comprises a recQ helicase domain is disclosed; furthermore expression vectors suitable for expression of said sequence in bacteria, plants, animals and fungi are disclosed; the invention refers also to organisms transformed by such vectors.

[0001] The present invention relates to the isolation and characterization of a Neurospora crassa gene encoding for an essential activity in the co-suppression process and to uses and applications thereof in vegetal, animal and fungine fields.

[0002] The production of transgenic organisms is of large utility both in basic and applied biological research. The transgenic DNA is usually integrated in the genome and transferred as a Mendelian character. However, in various instances, the transgene introduction induces gene silencing phenomena (Flavell, R. B. 1994), i.e. the repression of the expression of the transgene itself and/or of one or more endogenous homologous genes.

[0003] The gene silencing can act at two levels: transcriptional (trans-inactivation) where transgenes contain sequences homologous to the silenced gene promoter (Vaucheret, 1993); and post-transcriptional (co-suppression) which requires homologies between coding regions (Flavell, 1994; Stam et al., 1997; Baulcombe, 1996).

[0004] Generally the silencing induced by a transgene requires an almost complete sequence homology (from 70% to 100%) between transgene and silenced gene sequences (Elkind, 1990).

[0005] In the Neurospora crassa filamentous fungus, during the vegetative phase, the presence of transgenes induces a post-transcriptional gene silencing phenomenon, named “quelling” (Cogoni et al., 1996).

[0006] By using the al-1 gene (albino 1) (Schmidhauser et al., 1990) as silencing visual marker, many features of the phenomenon have been discovered (Cogoni et al., 1996). Particularly the al-1 gene “quelling” in Neurospora is characterized in that: 1) the gene silencing is reversible further to the loss of transgene copies; 2) the reduction of mRNA basal level results from a post-transcriptional effect; 3) transgenes containing at least a region of 132 base pairs which is identical to the region encoding for the target gene are sufficient to induce the “quelling”; 4) the duplication of promoter sequences is ineffective to induce the silencing; 5) the “quelling” exhibits a dominant behavior in eterocarions containing both transgenic and untransformed nuclei, indicating the involvement of a molecule which is acts “in trans” among the nuclei; 6) the expression of an aberrant RNA transcribed by the transgenic locus is strictly correlated to silencing, suggesting that the “quelling” can be induced and/or mediated by a transgenic RNA molecule.

[0007] Therefore homologies between Neurospora silencing and plant co-suppression can be pointed out. The gene silencing in Neurospora is reversible, as result of transgenic copies instability during mitotic phase; in plants also the co-suppression reversion is associated with the reduction of transgene copy number, resulting from intra-chromosomal recombination during mitosis or meiosis (Mittelstein Scheid et al., 1994; Stam et al., 1998). Thus both in plants and in Neurospora the transgene presence is required to maintain the silencing. As in Neurospora, a decrease of the mRNA basal level of the silenced gene results from a post-transcriptional mechanism (Dehio and Schell 1994; van Blokand et al., 1994; de Carvalho et al., 1995). Furthermore to induce the “quelling”, transgenes must contain a portion of the silencing target gene coding sequence, being the promoter region ineffective. In plants coding regions with no promoter sequences can induce silencing (van Blokand et al., 1994) and, as in the “quelling”, promoters or functionally active gene products are not required for the co-suppression.

[0008] One of the similarities between “quelling” and co-suppression in plants is that both mechanisms are mediated by diffusion factors. In Neurospora eterokaryotic strains, nuclei wherein the albino-1 gene is silenced are able to induce the al-1 gene silencing of the other not transformed nuclei, all sharing the same cytoplasmic environment (Cogoni et al., 1996). In plants the presence of a diffusion factor results from the fact that the co-suppression is effective in inhibiting the replication of Tobacco Etch Virus (TEV), a RNA virus with an exclusively cytoplasmic cycle. The occurrence of highly diffusible factors, which are effective to mediate the co-suppression, has been demonstrated using the grafting technique in tobacco (Palaqui et al., 1997), showing that silenced tobacco plants are able to transfer the silencing to non-silenced plants through grafting.

[0009] The fact that “quelling” and co-suppression share all these features suggests that mechanisms involved in post-transcriptional gene silencing in plants and in fungi can be evolved by an ancestral common mechanism.

[0010] Recently gene inactivation phenomena resulting from transgene introduction have been disclosed in animals. In Drosophila melanogaster the location of a transgene close to heterochromatic centers results in a variegate expression (Wallrath and Elgin, 1995; Pirrotta, V., 1997). Similar expression profiles have been observed when the reference transgene is within tandem arrayed transposons, indicating that tandem repeats are effective to induce the chromatin condensation. (Dorer and Henikoff, 1994). Again in Drosophila Pal-Bhadra et al. (1997) have observed that the transgene introduction can lead to gene inactivation phenomena, similar to the co-suppression.

[0011] Gene silencing phenomena resulting from transegene sequence repeats have been disclosed recently in mammalians.

[0012] Garrick et al. (1998) produced mouse transgenic lines wherein 100 transgenic copies are present only in a locus and are directly tandem arrayed. The transgene expression has been disclosed to be inversely proportional to the number of occurring copies, indicating that silencing phenomena dependent on repeat copies are present also in mammalians.

[0013] Therefore the identification of Neurospora genes which are involved in the silencing is the first step to modulate the same process in plants, animals and fungi. The silencing modulation is of great relevance when transgenic organisms able to express the desired phenotype are produced.

[0014] The authors of the present invention have already isolated Neurospora crassa strains having mutations regarding essential functions for gene silencing mechanism (Cogoni and Macino, 1997); 15 independent isolated mutants define three complementation groups, thus identifying the qde-1, qde-2 and qde-3 genes (qde stands for “quelling”-deficient), whose products are essential to the silencing machinery. qde genes are essential to the Neurospora silencing, as suggested by the fact that silencing of three independent genes (al-1, al-2 and qa-2) is impaired by qde mutations (Cogoni and Macino, 1997).

[0015] The authors of the invention have identified and cloned now one out of Neurospora qde genes, thus identifying one of required factors for silencing. By considering the similarity between “quelling” and co-suppression, genes orthologous to the isolated gene are involved in co-suppression and more generally in gene silencing in other organisms, like plants, fungi and animals.

[0016] The present invention can be applied with reference to two general scope: 1) silencing potentiation as a tool for inactivating more effectively and durably a desired gene, and 2) silencing suppression to obtain a better expression of the introduced transgenes.

[0017] As to the silencing potentiation, the over-expression of one or more genes controlling the phenomenon can lead to higher efficiency and/or stability thereof. Therefore the introduction of qde-3 gene or of homologous genes thereof in microorganisms can constitute a tool to repress more effectively gene functions. Particularly this approach is specially useful in plants wherein the co-suppression is usually used for the “knock-out” of gene functions. In plants again the gene silencing potentiation can be used to obtain lines resistant to pathogen virus, by introducing transgenes encoding for viral sequences, in order to achieve the expression inhibition of the virus itself (Flavell et al., 1994).

[0018] Analogous applications are suitable for animals, wherein some indications suggest that silencing can inhibit the suitable expression of introduced transgenes (Garrick et al., 1998).

[0019] On the contrary, there are instances wherein it is desirable not to have or to reduce the gene silencing, i.e. where a transgene is to be over-expressed. It is known that the co-suppression is strictly correlated both with the presence of an high copy number of the transgene, and with a transgene high expression. This correlation can hamper the production of transgenic organisms which express a transgene at high levels, because more high is the expression and/or the copy number, more probable is to evoke silencing responses. As above mentioned, analogous mechanisms of gene inactivation, dependent on a high copy number, have been disclosed in animals. In these circumstances plant or animal lines, totally or partially ineffective for silencing, constitute an ideal recipient wherein the desired gene can be over-expressed. The invention can be applied within this scope using different approaches:

[0020] A) Identification and Production of Mutant Lines in Genes Homologous to qde-3 Gene, in Plants, Animals and Fungi.

[0021] The knowledge of Neurospora qde-3 gene, essential for silencing mechanism, can allow the isolation of mutant lines in other organisms, mutated in genes homologous to qde-3. For example by means of amplifications using degenerated primers, designed from the most conserved regions of qde-3 gene, mutant lines in homologous genes can be identified, by analysis of insertion mutant gene banks, already available for many plant species. Both in fungi and animals such mutants can be obtained, following the identification of the homologous gene, by means of “gene disruption” techniques using homologous recombination.

[0022] B) Reduction of qde-3 Gene Expression

[0023] Other strategies for the production of silencing-deficient lines comprise the use of Neurospora qde-3 gene or homologous genes thereof. qde-3 or homologous genes can be introduced into suitable expression vectors to express them in an anti-sense orientation in order to inhibit the expression of resident endogenous genes. Alternatively portions of qde-3 or of homologous genes can be over-expressed, in order to obtain a negative dominant effect and thus blocking the function of qde-3 endogenous genes.

[0024] The authors of the present invention have cloned and characterised the Neurospora crassa qde-3 gene. The sequence analysis showed that qde-3 gene belongs to a highly conserved gene family, from E. coli to humans, named recQ. Genes belonging to this family encode for DNA helicase, as demonstrated by in vitro assays (Gray et al., 1997). The recQ helicase family is involved in recombinant processes. Mutations of these genes produce iper-recombinant phenotypes as, for example, the S. cerevisiae Sgs-1 gene involved both in meiotic and mitotic recombination.

[0025] The authors of the invention for the first time have demonstrated that a gene encoding for a recQ DNA-helicase is involved in gene silencing induced by transgenes. Therefore for the first time it is disclosed that a gene belonging to the recQ family, other than acts during recombination, is also an essential component of the inactivation of repeat sequences.

[0026] Therefore it is an object of the invention a nucleotide sequence encoding for a protein characterized in having a silencing activity and comprising a recQ helicase domain, wherein the domain is at least 30% homologous with the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1. More preferably said homology is of at least of 60%. Most preferably the recQ helicase domain comprises the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1. According to a particular embodiment the nucleotide sequence encodes for a protein having the amino acid sequence of SEQ ID No. 1, or functional portions thereof. Even more preferably the nucleotide sequence of the invention is the sequence of SEQ ID No. 1 or its complementary sequence.

[0027] A further object of the invention is an expression vector comprising, under the control of a promoter that is expressed in bacteria, the nucleotide sequence of the invention. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the invention in bacteria can be used and is within the scope of the invention.

[0028] A further object of the invention is an expression vector comprising, under the control of a promoter which is expressed in plants or in specific plant organs, the nucleotide sequence of the invention, both in a sense and anti-sense orientation. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the invention in plants or in specific plant organs can be used and is within the scope of the invention.

[0029] A further object of the invention is an expression vector comprising, under the control of a promoter which is expressed in fungi or in portions thereof, the nucleotide sequence of the invention, both in a sense and anti-sense orientation. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the invention in fungi or in portions thereof can be used and is within the scope of the invention.

[0030] A further object of the invention is an expression vector comprising, under the control of a promoter that is expressed in animals, the nucleotide sequence of the invention both in a sense and anti-sense orientation. Those skilled in the art will appreciate that any plasmid suitable for a correct and effective expression of the protein of the invention in animals can be used and is within the scope of the invention.

[0031] A further object of the invention is a prokaryotic organism transformed by using the expression vector active in bacteria of the invention.

[0032] A further object of the invention is a plant or a specific plant organ transformed by using the expression vector active in plants of the invention.

[0033] A further object of the invention is a plant mutated at the nucleotide sequence of the invention and having a reduced or inhibited silencing activity.

[0034] A further object of the invention is a fungus transformed with the expression vector of the invention active in fungi.

[0035] A further object of the invention is a fungus mutated at the nucleotide sequence of the invention and having a reduced or inhibited silencing activity.

[0036] A further object of the invention is a non-human animal transformed with the expression vector of the invention active in animals.

[0037] A further object of the invention is a non-human animal mutated at the nucleotide sequence of the invention and having a reduced or inhibited silencing activity.

[0038] A further object of the invention refers to a protein characterized in having a silencing activity and in comprising a recQ helicase domain, wherein the domain is at least 30% homologous to the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1. Preferably the recQ helicase domain is at least 40% homologous with the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1. More preferably the recQ helicase domain is at least 60% homologous with the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1. Most preferably the recQ helicase domain comprises the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1. According to a particular embodiment the protein comprises the amino acid sequence of SEQ ID. No.1 or functional portions thereof.

[0039] It is within the scope of the invention the use of the nucleotide sequence of the invention to modulate gene silencing in plants, animals and fungi.

[0040] It is within the scope of the invention the use of the nucleotide sequence of the invention to potentiate the antiviral-response in a plant.

[0041] The present invention now will be disclosed by way of non limiting examples with reference to the following figures:

[0042]FIG. 1: Southern blot analysis of genomic DNA extracted from (A): untransformed wild type strain, (B): 6xw recipient strain and (C): untransformed wild type strain, SmaI and HindIII digested, blotted and al-1 gene probe hybridized. The 3.1-Kb band corresponds to the endogenous al-1 gene, while the 5.5-Kb band corresponds to tandem arrayed al-1 transgenes. The larger band represents undigested methylated DNA.

[0043]FIG. 2: Linear map of the pMXY2 plasmid. Plasmid genes are shown as box. bmI: beta-tubulin allele which is responsible for benilate resistance; Amp: ampicillin resistance; qa-2 P: qa-2 gene promoter; TrpC T: trpC gene terminator. SphI and BglII are restriction sites used for the plasmid recovery from the 627 mutant chromosomal DNA.

[0044]FIG. 3: Schematic representation of pQD6 and pQ35 plasmids. Restriction sites (BglII for pQD6 and SphI for pQ35) used for the recovery of the chromosomal DNA of the 627 strain are reported. Chromosomal sequences, flanking the integration site, are represented as segments. Restriction sites used to isolate DNA fragments used for probing the gene library are also represented.

[0045]FIG. 4: Nucleotide sequence of the 6.9-Kb fragment containing the qde-3 gene and flanking sequences. The amino acid sequence is shown above the nucleotide sequence. The bold sequences represent two introns of 98 and 68 nt. In these regions the underlined nucleotides identify consensus sequences of the donor site, the acceptor site and the internal sequence or lariat. It is also represented the pMXY2 plasmid insertion site, in the 627 mutant, used for insertional mutagenesis. The portion encoding for the helicase domain is underlined.

[0046]FIG. 5: Nucleotide sequence (SEQ ID No. 1) of the encoding portion reported in FIG. 4 and deduced amino acid sequence. Amino acids from 897 to 1330, which define the recQ DNA-helicase domain, are underlined.

[0047]FIG. 6: Multiple alignment, at the conserved domains, among qde-3 and other proteins belonging to recQ family. arab recQ: A. thaliana isologous; E. coli recQ; S. pombe hus-2; S. cerevisiae sgs-1; human wrn: Werner syndrome; human blm: Bloom syndrome. Identical amino acids are shown in bold.

[0048] Materials and Methods

[0049]E. coli Strains

[0050]E. coli strain HB101 (F^(—), hsdS20(rb^(—), mb^(—)), supE44, recA13, ara14, proA2, rspL20(str^(r)), xyl-5) was used for cloning.

[0051]Neurospora crassa Strains and Growing Conditions

[0052]Neurospora crassa following strains, supplied by Fungal Genetic Stock Center (FGSC, Dpt. Of Microbiology, University of Kansas Medical Ctr. Kansas City, KA) were used:

[0053] Wild type (FGSC 987);

[0054] qa-2/aro9 (FGSC 3957A), (FGSC 3958a).

[0055] The 6XW strain (Cogoni et al., 1996) was obtained upon transformation of the FGCS 3958a strain with pX16 (Cogoni et al., 1996). This plasmid contains the qa-2 gene used as selective marker and the al-1 coding sequence.

[0056] The mutated strains M7, M20 (qde-1); M10, M11 (qde-2); M17, M18 (qde-3) are described in Cogoni and Macino, 1997.

[0057] The qde mutants were obtained by UV mutagenesis. As recipient the transforming strain (6xw) silenced at the albino-1 gene was used. qde mutants were selected for their ability to recover a wild type unsilenced phenotype and then classified in three different complementation groups. By analyzing the al-2 gene quelling frequency all of qde used mutants are defective for the general silencing mechanism.

[0058] Complementation assays with not forced heterocaryons were carried out according to Davis and DeSerres, 1970.

[0059] Plasmids and Libraries

[0060] The plasmid pMXY2, disclosed in Campbell et al., used for insertional mutagenesis was obtained from FGSC. The plasmid contains the Bm1 gene (allele responsible of the benilate drug resistance), that was used as selective marker after transformation. The genomic DNA containing the qde-2 gene was isolated from a N. Crassa gene library in cosmids. (Cabibbo et al., 1991).

[0061]N. crassa Transformation

[0062] Spheroplasts were prepared according to the Akins and Lambowitz (1985) protocol.

[0063] Southern Blot Analysis

[0064] Chromosomal DNA was prepared as disclosed by Irelan et al., 1993. 5 μg of genomic DNA were digested and blotted as reported in Maniatis et al.

[0065] DNA probes were: a) as to the al-1 gene the probe is represented by a XbaI-ClaI restriction fragment of pX16 (Cogoni et al., 1996); b) as to the BmI gene the probe is represented by the 2.6 Kb SalI fragment of pMXY2.

[0066] Northern Blot Analysis

[0067]N. crassa total RNA was extracted according to the protocol described by Cogoni et al., 1996. The mycelium was grown for two days at 30° C., then powdered in liquid nitrogen before RNA extraction. For Northern analysis 10 μg of RNA were formaldehyde denatured, electrophoresed on a 1% agarose, 7% formaldehyde gel, and blotted over Hybond N (Amersham) membranes. Hybridization was carried out in 50% formamide in the presence of 32 labeled DNA probe 1.5×10⁶ cpm/ml.

[0068] Results

[0069] Isolation of Silencing Mutant by Insertional Mutagenesis

[0070] Neurospora strain (6XW) wherein the albino-1 resident gene was steadily silenced was UV mutagenised, and qde (“quelling” deficient) mutants were isolated (Cogoni and Mancino 1997). The 6XW strain shows an albino phenotype due to the lack of carotenoid biosynthesis, as results by the silencing of the albino 1 gene expression (Schmidhauser et al., 1990). A mutation interfering with the silencing machinery is easily detectable by producing a wild type phenotype (bright orange) of the carotenoid biosynthesis. By means of complementation assays it was possible to establish that qde mutants belong to three complementation groups, indicating the presence of three genetic loci involved in the Neurospora silencing mechanism. In order to isolate the qde genes an insertional mutagenesis was carried out with the 6XW strain, previously used for UV mutagenesis. The insertional mutagenesis was carried out by transforming the 6XW strain with a plasmid, taking advantage of the fact that, after the transformation, plasmids are randomly inserted in the Neurospora crassa genome. The mutagenesis was carried out transforming the 6XW silenced strain with pMXY2 (see Materials and Methods) which contains the benilate resistance as selective marker. Transformed strains able to grow in the presence of benilate containing medium and showing a wild type phenotype for the carotenoid biosynthesis were selected. Out of 50.000 isolated independent transformed strains, a benilate resistant strain (627) was isolated, which showed the bright orange phenotype expected for a qde gene mutation. In order to verify that the silencing release was effectively due to a qde gene mutation and not to the loss of al-1, the genomic DNA of the strain 627 was extracted and digested with SmaI and HindIII restriction enzymes. After blotting, DNA was hybridized with a probe corresponding to the coding sequence of al-1. The SmaI site is present only once in the al-1 transgene containing plasmid and the digestion by using said enzyme produces a 5.5 Kb fragment corresponding to tandem arrayed al-1 transgenes, while a 3.1 Kb fragment is expected from the resident al-1 locus. FIG. 1 shows that the number of al-1 transgenic copies present in the 627 strain is comparable to that present in the silenced 6XW strain.

[0071] The 627 Strain Includes a Mutated qde3 Gene

[0072] The 627 strain was assayed in a heterokaryon assay with a wild type strain and with M7, M20 (qde-1) M10, M11 (qde-2) mutants (Cogoni and Macino, 1997). As shown in Table 1 the al-1 gene silencing is restored producing an albino phenotype in all of heterocaryons but M17 and M18. This behavior is consistent with the presence of a qde-3 gene recessive mutation in the 627 strain. TABLE 1 Reciprocal heterokaryons among 627 mutant and previously characterized qde mutants. 627 M7 M20 M10 M11 M17 M18 627 WT AL AL AL AL WT WT M7 WT WT AL AL AL AL M20 WT AL AL AL AL M10 WT WT AL AL M11 WT AL AL M17 WT WT M18 WT

[0073] Recovery of Sequences Flanking the PMXY2 Plasmid Integration Site

[0074] In order to recover sequences flanking the integration site or sites the following methodology was carried out. The 627 strain genomic DNA was restricted with SphI and BglII enzymes. As shown in the map of FIG. 2 the enzymes digest respectively upstream and downstream to the region containing both the ampicillin resistance gene and the origin of replication present in pMXY2. Subsequently the genomic DNA was ligated and the product used to transform E. coli cells. The screening was performed in an ampicillin-containing medium. pQD6 and pQ35 plasmids were recovered from BglII and SphI restricted chromosomal DNA, respectively (see FIG. 3). Two DNA fragments containing sequences flanking the integration site were isolated by using, respectively, BglII and SalI enzymes for pQD6, and SphI and HindIII enzymes for pQ35 (FIG. 3).

[0075] Isolation of Genomic Clones, their Subcloning and Complementation of the qde-3 Mutant

[0076] The two fragments from pQD6 and pQ35 plasmids were used to probe a Neurospora crassa genomic library in cosmids. Cosmids 6E8 and 54D7, both containing about 30 Kb genomic DNA inserts, were isolated. Both the probes recognize the same cosmids, thus indicating that the two flanking sequences are contiguous. Cosmids 6E8 and 54D7 were used in transformation experiments with M17 and M18 mutants. Both of cosmids are able to restore the al-1 gene silencing in the two mutants, determining an albino phenotype. Furthermore the introduction of same cosmids into the M10 (qde-2) or the M20 mutant (qde-1) is not effective to restore the silencing.

[0077] The 6E8 cosmid was used to subclone a 9 Kb SphI-SphI fragment. This subclone was used for transformation experiments and resulted to be able to complement the qde-3 phenotype, indicating that a qde-3 functional gene is present in this plasmid.

[0078] Isolation and Sequence of the qde-3 cDNA

[0079] The SphI-SphI region was sequenced, like the corresponding cDNA, by using RT-PCR. The latter sequence was used to deduce the qde-3 amino acid sequence and map the introns therein. The qde-3 gene encodes for a 1900 aa. putative protein (200 KDa). The genomic clone contains two introns of 98 nt. and 68 nt., respectively. Intron acceptor and donor sequences were identified and correspond to described consensus sequences (FIG. 4). Furthermore the pMXY2 plasmid insertion site within the gene in the 627 transforming strain is indicated. The insertion site was deduced by analysis of pQD6 and pQ35 plasmid sequences.

[0080] The cDNA sequence is shown in FIG. 5 (SEQ ID No. 1), wherein the helicase domain containing 434 amino acids from 897 aa to 1330 aa is underlined.

[0081] The qde-3 Gene is Belonging to recQ Helicase DNA Family

[0082] The 1900 aa sequence was used to search in database of amino acid sequences, by using the BLASTP algorithm. Significant homologies were identified with 6 genes belonging to the reQ family, belonging to the helicase group containing the DEAH consensus sequence. FIG. 6 shows the homologous region sequence alignment of helicase domains, as defined in FIG. 5, among qde-3 and genes belonging to recQ helicase family. qde-3 shows the highest homology with hus-2 (55% amino acid identity) and the lowest homology with Wrn (40% identity).

[0083] Plant Expression Vector

[0084] The qde-3 gene was inserted, in a sense orientation, into a vector containing a plant expression “cassette”, including the 35S promoter and the PI-II “terminator” sequences. The vector also includes the Streptomyces hygroscopicus bar gene, which confers the phosphinotricine herbicide resistance to transformed plants. In an analogous vector, qde-3 was inserted in an anti-sense orientation with respect to the 35S promoter.

[0085] The obtained vectors can be utilized to over-express the qde-3 gene in plants, or to repress the gene expression of resident genes, which are homologous to qde-3, respectively.

[0086] Fungus Expression Vector

[0087] The qde-3 gene was inserted in a vector containing a fungal specific expression “cassette”, comprising the A. nidulans trpC gene promoter and terminator, both in a sense and an anti-sense orientation. In addition the vector contains the bacterial hph gene, which confers the hygromicine drug resistance. The sense plasmid can be used to over express the qde-3 gene, whereas the anti-sense plasmid is used to repress the expression of qde-3 homologous genes in various fungine species.

[0088] Mammalian Expression Vector

[0089] The qde-3 gene was inserted in a vector containing a mammalian specific expression “cassette”, including the cytomegalovirus (CMV) promoter and SV40 termination and polyadenylation sequences both in a sense and anti-sense orientation. The vector includes also the neomicine phototransferase gene, as marker for mammalian cell selection. The sense plasmid can be used to over express the qde-3 gene, whereas the anti-sense plasmid can be used to repress the expression of qde-3 homologous genes in various mammalian species.

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1 3 1 6916 DNA Neurospora crassa 1 ccaccaccac caccaccacc acaacaacca actcaacaac gaactcaaca accaacccca 60 aacccaacct cgacctcaac ctcaacctca acccttgcga cctcgagata cacaaacaca 120 tcctcgacaa atgacgcccg acccgctaca cgccaacaga ttgcccccga ggtaggagca 180 tcgacgcatc aggattcagt tggacttgga gaaggaggag gaggaggaat ggcgaagctc 240 tcagtcaaga acaacctgcc acggccgcac ttggtctcct tgtcgtcgtc aacgacaggc 300 tctgggtctg gttctgcgtc taggtcagct tctgctaagc acggaagtgc cggttccagt 360 acctttgatc atgaacaaca tcaacaacat caacaacaac aacaacaaaa gcgccagcgg 420 tcgcaatcag aagcacgaca acagcagcag caacagcaac agcaacagca acagcaacaa 480 caacaacaac aagcacagca ccatgcacat tctacatatg cacaaagacc ccaacccacc 540 ccccaacaac gaccacccca aaacctactg acacctgctt caaccactgg tgccagcgtc 600 ggcccgctcc aacgcgcata ctcggtttca ttagctgcga gacagtcccc ctcgacaaac 660 ttggtccgtc caaagaccga ctcgccagct ccccacactt tacacctcaa gaacaagaag 720 aacctccgtc accccgcccc cacgcccgac agtccgatcg tagacgacga tattttctcc 780 gacgccgtcg atcttaccga agaactcgat catgaccatg atctcaacgg caaagacaaa 840 gacaacaccg acaacgacaa cacagtcgct tccagttcgc taatagggtt cggcgatgac 900 aagttactgt ggcgagagga ctttgctgag cgtgcagagc ccgaacatga aagaggtggg 960 agcaggcctc gccaggtcaa gaaacggaag atatcgaatg actacattat gaaggatgag 1020 gatgtctcgc tttttgatga tgatggcgag gaggacgagt ttatggatat caatgagcta 1080 gttcaggggg atcgggaaag tactccgaag ccaaaggcta catcgaggtc tgtctcgacg 1140 aggctgccgc ctacagtatc gctgcaacgg ggtcggtctc ctaagaggaa ggaggcttca 1200 gttgaaaagc gcacaacgga aaaccagcaa caggctgaca gagaagacga accgtcgttt 1260 atgtcaagtc cagatgtcga caactcccgc aagcgaaagt cttctggatc gcccacaggt 1320 ttaacgacgc caagacccca gcagaagcaa acggaagagg tcccaggtac gaccaccgcc 1380 aagaagccac ggcgcagtga agtgatggac tcggaggacg aggcattcac tcctctttct 1440 gctgggtcgc tgcctgggag tgcggagttc ttcagaagcg gtgggaccac cacacgggaa 1500 ttgggtttgg acgaagacac ggttatggac acgcctagta ggccaccggt cgagtccact 1560 ttgccaactc tcgagtctgt ggaaagtcga ccaccccccc tgccgcccat ggatctacca 1620 tcacagcgaa aaccgctaga gccgttgaac actccgcgca accagctgct tgagtcggtc 1680 gaaaggccaa cacagcagcc gtcggtgggg ccgagttttg cacagagtag cacactcgcc 1740 gaaagctccc tgccgccgtc aatgccgccg ccaagtgaag accccctcaa caccagggag 1800 aacagcaacc ttgaggagtt cgactacaag ctttacaaac ccctgctaga tcttttcgtc 1860 aacgcacccg caatcttgga aagagaactg agcgccgtta atgacgagct tcaggagaac 1920 atgatcaagc tgcgggactg tctgcgcctg cccagggaag aaagagacag ggcacgcgaa 1980 gaggtgaaga aggaaaagga aatgctcaag cgacgggaca ttgcgctcag agccctccag 2040 gacgaacaca agttgtacgt caagaaacgc aaagagcata atttgatcaa cgaggaaatc 2100 gttcgcgctt atgctgaaga agacgatgag tacgaggatc agttaatggc gcagctggac 2160 aagttggatg atgaggttga ggctatcgta aagagtctga caaggcttat tgtggcggcg 2220 gggatcacgg agaagagctt tgacctaaag aaggaggagg aagaggagga ggagaagccg 2280 atcatcatag cgactccgac accttcgacg aggaccgagg ccccggttct gccgacgacc 2340 gagtatcata attcccagca ggtcatattg cagactcaac atcctgctgc gcagcaggtt 2400 tctcaccggg tgccaccacc tccgacaccg agttttcaaa cggcgcgcca gactccggtg 2460 tcatatcaga gcagaccgac caacaactcc tttcctgata tctcggcgga agaagccatg 2520 atgttcgata aagaagaccc cttcatggaa caacagcacg ccccggcctc tgctcccttc 2580 caggccaccc ttccccagcg caacagccct ttcaaaaccg ccccgttcaa gccagtccac 2640 ggccacgatt actttgacga tgaagacgac gatgccgacc tcctggcagc agtagacagc 2700 gccgagacgt atacttctac ggccgccacc accaccacca acaacaacaa tcacttacga 2760 tcacaatcgg tgatgtcaac atccacggcg accacgatca aaccgaggaa acgcaacgaa 2820 aatgccaatg ccaagaagcc caagtccgta catgcaaagt tatcgatgcc gcccgaaaag 2880 atgaagtatg cgtggtcgaa tgatgtgagg aaggctctca aggataggtt tcggatgtcg 2940 gggttcagac agaatcagtt ggaggctatt aatgctactt tgggtggtaa ggtgagttct 3000 ctgtccttta cctatctggg agagaccaag aaggagagag agagagagag gaggggaaga 3060 cgaaaatgga ctttgctgac tctagaaagg atgcctttgt gttgatgccg actggtggtg 3120 gaaagtctct gtgctatcag ttgccggctg tagtcaggag cggcaagacg cgtggtatca 3180 cagtcgtcat ctcccctctg ctaagtctga tgctggatca agtcaaccat ttggcaaacc 3240 tgatgatcca agcttacgct ttcaacggag acatgaactc agaaatgcgc cgaatggtgt 3300 ttcagaagct tgatgctgag catcctgagc atgagctcca actgctctat gtcaccccgg 3360 agatggtgag caagaaccag acattcgtca acaagatgat ggacctctac cggaggaaaa 3420 agctggctag aattgttatc gacgaggctc actgcgtcag tcaatggggc catgacttcc 3480 gacccgatta caaagctata ggagagtttc gtaagaggtt tcccggagtt ccggtcatgg 3540 ctttgacagc gacggcaaca cagaacgtca tcttggatgt caagcataac ctggcaatgg 3600 aggactgcca gactttctcc cagagcttta atcggccgaa cctctactat gaggtcagga 3660 tgaaggagca gaatctgatt gcccgcatcg cagagttgat caaggagaag tatgacggcc 3720 agacgggtat catctacaca ttatcaagaa agagtgccga gaacatcgcc aaaaatctcc 3780 aggaaaaaca ccgcatcaaa gcaaagcact accatgcctc catcaccacc gacgaaaaga 3840 tcagcgttca acatgaatgg caaaccggcc gagtcaaagt cgtggtagcc accattgcct 3900 tcggcatggg catcgacaag cctgacgtcc gctttgttat ccaccagcac atccccaagt 3960 cgctcgaagg ttactatcaa gaaaccggcc gcgccggacg tgacggcaag ccatcggact 4020 gctacttgta ctttgcctat ggcgacattc aatccctacg tcgtatgatc gccgacggcg 4080 aaggtgacta cgcgcaaaag gagcgtcagc tacaaatgct caaccgtgtg gtcagctatt 4140 gcgagtcgca gcacacgtgc cggcgcgaag aagtgctccg ctactttggc gaggagtttg 4200 actaccggaa gtgtagagac ggatgcgata actgccggaa cggacgcatc tcgaagtcga 4260 cggagatgag ggattttacg gaaatcgcct tcgccgcgat cgaggtggtg aagagccagc 4320 agcccatcac gctgggcaag ctgtgcgaca tcctgatggg caagagaaag aacgagcacg 4380 gtggcgtgtg tcactttggt atcgccaagg ggagcacgca gagggagctg cagaggatcg 4440 tgctgcagct gaatttccac aaggcgctgg gcgaggacaa tatcatgaat ggggcgggga 4500 tgcctattac ctactatatt gtgagtgctg tcccggttgg tcttgcatat ctggctttgt 4560 tgctttgcta acacagcagc tcgtacagac cggccctgaa gctggtgctt acctctacaa 4620 tggcaagcgg ttgatgctgc cagttccctc aaacaagtcc gtcgaacccc cgtctcggtc 4680 taagcagcgg agccgtcgag tcgatgagga tatggatgag caagaacttt ccaccctgca 4740 acgaccgcca acatcaacaa atgtctcttc acccgttcga gccaccaaga aacgaagttc 4800 caaaaaggct ttaccgaccc tcatcgccga ctacgaagag cccagctccg acggtcctca 4860 cggtcctctc cacgccaacg gctatgagcg cgataacttt gtcgtatccg ataatgttga 4920 acccgaagag gaagaagatg ccttcgaacc tgtccgcccc tcgcggcgcg gcccatcttc 4980 tcgcgctacc cgccctcaac accgccagac caccctttat gacaccctct cccacaccca 5040 acaatcccaa accgtctccc aacacctcgc cactttgggt ccgcccatcg acgcccgcac 5100 catgcataac ccccgctacg cccagcttga cgaggtccac caggatattg tcgatgcctt 5160 tgttgaagaa gtcaaggtct tcgaggagga ctttcgcaac aggaaccaca tgcgcaaacc 5220 catctttacc gagacgcagt accgtgagat ggcaatccgg tggacgcggt cgttagacgc 5280 gatgcgcgcg atcccggata tcaaccagga taaagtagat cggtatggtg ccaaattcat 5340 cccacttgtg gagcggttct gggggaatta tcaggagatg atggggggag ggtatgataa 5400 tcctgctgtg gctggcgatg aggatgatga tgagggcccc aggaggacag gaaatggaaa 5460 aggggggaat aagaagggag gaggaggagg aggaggaaat gaagtagtgg atttgattag 5520 tagtgatgag gatgaacctc cggctcgtgc accatcgcgg aatgcggggc gaggaaaggc 5580 acagtcgaca cgtgggggac aaatccaaga taaaggccga gcagtcaacc gccgcggaga 5640 acccatcgcc gaagaagacg aagaagacta cgggctaagc gaccccgata tcgacgccat 5700 cgatccagac gccatcaccg cctccgacaa ctccgacgaa gaagatgatg atgatgatga 5760 cgaagacctc gaatcctccc gctacttctc cggctcaaca ggcccgcccg tctccaaagc 5820 cgtgcaggat gctcgactcc gtgaacaact ttccatgtac gcctccggcg gcagctcttc 5880 gaaaggtagc tacggctcag ggcgcgcatc aggaggatct tcgtcgagag cgtcgggatc 5940 aggatggaga ggtggaggag caggtgggaa gaaatactac aggaagaaga gggctggttc 6000 ttcggctgct ggtggtggtg gtgcaggagg agggggagtt acaaaacgga aggcgagtgg 6060 gagtggcgcg aagacggcga ggaagagggg tgcatctact gcgccgaaga caacgacgag 6120 agggggagga tctggagctg ggtctagagg aggcggtgct ggtggtgctg gtggtgctgg 6180 tgctggtgct ggtgctggag gagggaaaag gggtggtgga ggtggaggag gaatgggagg 6240 gataagtgtt atgcctcatt agctatttta tagcatatcg catttataca gtgtcttatg 6300 gaagggagga ggagaagaag aaggataagc tggcataagc ttgaaccggc caggccaaaa 6360 tggccagaga gctcaccggg caatcgagct tgaaatgagc ttgacatatt aggtattccc 6420 gagaatatag cgggattaca aggcacttac tttaccaagt cgaaagggac gagccaaatc 6480 tatggtactc gccagttgcg caacgttgag ttttatcatt cgtggagttt tcatcgtgga 6540 gtttttatta tcaactattc gttgtatagt tttcgttgta gatgttagtt ccggacgatc 6600 aaaaggggaa gtgtggaaca gagaagtcga aaggacaagc caaaatgaca tggcagtgtc 6660 cagtcagata ccctccagac aaaaccagac accaataaca aacccttcaa ccataacacc 6720 agcaaagcca atccttaggt acctacctag ggtagggtag gtccaggaat gtcttcccca 6780 aaggtacctc tacttattca tgttacgctc catcagtccc atcgcttagc atcgctgccc 6840 ggttacctat ctctacctct acctctacct ctacctctac ctctacctct acctctatct 6900 ctacctctac ctctac 6916 2 5868 DNA Neurospora crassa CDS (1)..(5868) 2 atg gcg aag ctc tca gtc aag aac aac ctg cca cgg ccg cac ttg gtc 48 Met Ala Lys Leu Ser Val Lys Asn Asn Leu Pro Arg Pro His Leu Val 1 5 10 15 tcc ttg tcg tcg tca acg aca ggc tct ggg tct ggt tct gcg tct agg 96 Ser Leu Ser Ser Ser Thr Thr Gly Ser Gly Ser Gly Ser Ala Ser Arg 20 25 30 tca gct tct gct aag cac gga agt gcc ggt tcc agt acc ttt gat cat 144 Ser Ala Ser Ala Lys His Gly Ser Ala Gly Ser Ser Thr Phe Asp His 35 40 45 gaa caa cat caa caa cat caa caa caa caa caa caa aag cgc cag cgg 192 Glu Gln His Gln Gln His Gln Gln Gln Gln Gln Gln Lys Arg Gln Arg 50 55 60 tcg caa tca gaa gca cga caa cag cag cag caa cag caa cag caa cag 240 Ser Gln Ser Glu Ala Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 caa cag caa caa caa caa caa caa gca cag cac cat gca cat tct aca 288 Gln Gln Gln Gln Gln Gln Gln Gln Ala Gln His His Ala His Ser Thr 85 90 95 tat gca caa aga ccc caa ccc acc ccc caa caa cga cca ccc caa aac 336 Tyr Ala Gln Arg Pro Gln Pro Thr Pro Gln Gln Arg Pro Pro Gln Asn 100 105 110 cta ctg aca cct gct tca acc act ggt gcc agc gtc ggc ccg ctc caa 384 Leu Leu Thr Pro Ala Ser Thr Thr Gly Ala Ser Val Gly Pro Leu Gln 115 120 125 cgc gca tac tcg gtt tca tta gct gcg aga cag tcc ccc tcg aca aac 432 Arg Ala Tyr Ser Val Ser Leu Ala Ala Arg Gln Ser Pro Ser Thr Asn 130 135 140 ttg gtc cgt cca aag acc gac tcg cca gct ccc cac act tta cac ctc 480 Leu Val Arg Pro Lys Thr Asp Ser Pro Ala Pro His Thr Leu His Leu 145 150 155 160 aag aac aag aag aac ctc cgt cac ccc gcc ccc acg ccc gac agt ccg 528 Lys Asn Lys Lys Asn Leu Arg His Pro Ala Pro Thr Pro Asp Ser Pro 165 170 175 atc gta gac gac gat att ttc tcc gac gcc gtc gat ctt acc gaa gaa 576 Ile Val Asp Asp Asp Ile Phe Ser Asp Ala Val Asp Leu Thr Glu Glu 180 185 190 ctc gat cat gac cat gat ctc aac ggc aaa gac aaa gac aac acc gac 624 Leu Asp His Asp His Asp Leu Asn Gly Lys Asp Lys Asp Asn Thr Asp 195 200 205 aac gac aac aca gtc gct tcc agt tcg cta ata ggg ttc ggc gat gac 672 Asn Asp Asn Thr Val Ala Ser Ser Ser Leu Ile Gly Phe Gly Asp Asp 210 215 220 aag tta ctg tgg cga gag gac ttt gct gag cgt gca gag ccc gaa cat 720 Lys Leu Leu Trp Arg Glu Asp Phe Ala Glu Arg Ala Glu Pro Glu His 225 230 235 240 gaa aga ggt ggg agc agg cct cgc cag gtc aag aaa cgg aag ata tcg 768 Glu Arg Gly Gly Ser Arg Pro Arg Gln Val Lys Lys Arg Lys Ile Ser 245 250 255 aat gac tac att atg aag gat gag gat gtc tcg ctt ttt gat gat gat 816 Asn Asp Tyr Ile Met Lys Asp Glu Asp Val Ser Leu Phe Asp Asp Asp 260 265 270 ggc gag gag gac gag ttt atg gat atc aat gag cta gtt cag ggg gat 864 Gly Glu Glu Asp Glu Phe Met Asp Ile Asn Glu Leu Val Gln Gly Asp 275 280 285 cgg gaa agt act ccg aag cca aag gct aca tcg agg tct gtc tcg acg 912 Arg Glu Ser Thr Pro Lys Pro Lys Ala Thr Ser Arg Ser Val Ser Thr 290 295 300 agg ctg ccg cct aca gta tcg ctg caa cgg ggt cgg tct cct aag agg 960 Arg Leu Pro Pro Thr Val Ser Leu Gln Arg Gly Arg Ser Pro Lys Arg 305 310 315 320 aag gag gct tca gtt gaa aag cgc aca acg gaa aac cag caa cag gct 1008 Lys Glu Ala Ser Val Glu Lys Arg Thr Thr Glu Asn Gln Gln Gln Ala 325 330 335 gac aga gaa gac gaa ccg tcg ttt atg tca agt cca gat gtc gac aac 1056 Asp Arg Glu Asp Glu Pro Ser Phe Met Ser Ser Pro Asp Val Asp Asn 340 345 350 tcc cgc aag cga aag tct tct gga tcg ccc aca ggt tta acg acg cca 1104 Ser Arg Lys Arg Lys Ser Ser Gly Ser Pro Thr Gly Leu Thr Thr Pro 355 360 365 aga ccc cag cag aag caa acg gaa gag gtc cca ggt acg acc acc gcc 1152 Arg Pro Gln Gln Lys Gln Thr Glu Glu Val Pro Gly Thr Thr Thr Ala 370 375 380 aag aag cca cgg cgc agt gaa gtg atg gac tcg gag gac gag gca ttc 1200 Lys Lys Pro Arg Arg Ser Glu Val Met Asp Ser Glu Asp Glu Ala Phe 385 390 395 400 act cct ctt tct gct ggg tcg ctg cct ggg agt gcg gag ttc ttc aga 1248 Thr Pro Leu Ser Ala Gly Ser Leu Pro Gly Ser Ala Glu Phe Phe Arg 405 410 415 agc ggt ggg acc acc aca cgg gaa ttg ggt ttg gac gaa gac acg gtt 1296 Ser Gly Gly Thr Thr Thr Arg Glu Leu Gly Leu Asp Glu Asp Thr Val 420 425 430 atg gac acg cct agt agg cca ccg gtc gag tcc act ttg cca act ctc 1344 Met Asp Thr Pro Ser Arg Pro Pro Val Glu Ser Thr Leu Pro Thr Leu 435 440 445 gag tct gtg gaa agt cga cca ccc ccc ctg ccg ccc atg gat cta cca 1392 Glu Ser Val Glu Ser Arg Pro Pro Pro Leu Pro Pro Met Asp Leu Pro 450 455 460 tca cag cga aaa ccg cta gag ccg ttg aac act ccg cgc aac cag ctg 1440 Ser Gln Arg Lys Pro Leu Glu Pro Leu Asn Thr Pro Arg Asn Gln Leu 465 470 475 480 ctt gag tcg gtc gaa agg cca aca cag cag ccg tcg gtg ggg ccg agt 1488 Leu Glu Ser Val Glu Arg Pro Thr Gln Gln Pro Ser Val Gly Pro Ser 485 490 495 ttt gca cag agt agc aca ctc gcc gaa agc tcc ctg ccg ccg tca atg 1536 Phe Ala Gln Ser Ser Thr Leu Ala Glu Ser Ser Leu Pro Pro Ser Met 500 505 510 ccg ccg cca agt gaa gac ccc ctc aac acc agg gag aac agc aac ctt 1584 Pro Pro Pro Ser Glu Asp Pro Leu Asn Thr Arg Glu Asn Ser Asn Leu 515 520 525 gag gag ttc gac tac aag ctt tac aaa ccc ctg cta gat ctt ttc gtc 1632 Glu Glu Phe Asp Tyr Lys Leu Tyr Lys Pro Leu Leu Asp Leu Phe Val 530 535 540 aac gca ccc gca atc ttg gaa aga gaa ctg agc gcc gtt aat gac gag 1680 Asn Ala Pro Ala Ile Leu Glu Arg Glu Leu Ser Ala Val Asn Asp Glu 545 550 555 560 ctt cag gag aac atg atc aag ctg cgg gac tgt ctg cgc ctg ccc agg 1728 Leu Gln Glu Asn Met Ile Lys Leu Arg Asp Cys Leu Arg Leu Pro Arg 565 570 575 gaa gaa aga gac agg gca cgc gaa gag gtg aag aag gaa aag gaa atg 1776 Glu Glu Arg Asp Arg Ala Arg Glu Glu Val Lys Lys Glu Lys Glu Met 580 585 590 ctc aag cga cgg gac att gcg ctc aga gcc ctc cag gac gaa cac aag 1824 Leu Lys Arg Arg Asp Ile Ala Leu Arg Ala Leu Gln Asp Glu His Lys 595 600 605 ttg tac gtc aag aaa cgc aaa gag cat aat ttg atc aac gag gaa atc 1872 Leu Tyr Val Lys Lys Arg Lys Glu His Asn Leu Ile Asn Glu Glu Ile 610 615 620 gtt cgc gct tat gct gaa gaa gac gat gag tac gag gat cag tta atg 1920 Val Arg Ala Tyr Ala Glu Glu Asp Asp Glu Tyr Glu Asp Gln Leu Met 625 630 635 640 gcg cag ctg gac aag ttg gat gat gag gtt gag gct atc gta aag agt 1968 Ala Gln Leu Asp Lys Leu Asp Asp Glu Val Glu Ala Ile Val Lys Ser 645 650 655 ctg aca agg ctt att gtg gcg gcg ggg atc acg gag aag agc ttt gac 2016 Leu Thr Arg Leu Ile Val Ala Ala Gly Ile Thr Glu Lys Ser Phe Asp 660 665 670 cta aag aag gag gag gaa gag gag gag gag aag ccg atc atc ata gcg 2064 Leu Lys Lys Glu Glu Glu Glu Glu Glu Glu Lys Pro Ile Ile Ile Ala 675 680 685 act ccg aca cct tcg acg agg acc gag gcc ccg gtt ctg ccg acg acc 2112 Thr Pro Thr Pro Ser Thr Arg Thr Glu Ala Pro Val Leu Pro Thr Thr 690 695 700 gag tat cat aat tcc cag cag gtc ata ttg cag act caa cat cct gct 2160 Glu Tyr His Asn Ser Gln Gln Val Ile Leu Gln Thr Gln His Pro Ala 705 710 715 720 gcg cag cag gtt tct cac cgg gtg cca cca cct ccg aca ccg agt ttt 2208 Ala Gln Gln Val Ser His Arg Val Pro Pro Pro Pro Thr Pro Ser Phe 725 730 735 caa acg gcg cgc cag act ccg gtg tca tat cag agc aga ccg acc aac 2256 Gln Thr Ala Arg Gln Thr Pro Val Ser Tyr Gln Ser Arg Pro Thr Asn 740 745 750 aac tcc ttt cct gat atc tcg gcg gaa gaa gcc atg atg ttc gat aaa 2304 Asn Ser Phe Pro Asp Ile Ser Ala Glu Glu Ala Met Met Phe Asp Lys 755 760 765 gaa gac ccc ttc atg gaa caa cag cac gcc ccg gcc tct gct ccc ttc 2352 Glu Asp Pro Phe Met Glu Gln Gln His Ala Pro Ala Ser Ala Pro Phe 770 775 780 cag gcc acc ctt ccc cag cgc aac agc cct ttc aaa acc gcc ccg ttc 2400 Gln Ala Thr Leu Pro Gln Arg Asn Ser Pro Phe Lys Thr Ala Pro Phe 785 790 795 800 aag cca gtc cac ggc cac gat tac ttt gac gat gaa gac gac gat gcc 2448 Lys Pro Val His Gly His Asp Tyr Phe Asp Asp Glu Asp Asp Asp Ala 805 810 815 gac ctc ctg gca gca gta gac agc gcc gag acg tat act tct acg gcc 2496 Asp Leu Leu Ala Ala Val Asp Ser Ala Glu Thr Tyr Thr Ser Thr Ala 820 825 830 gcc acc acc acc acc aac aac aac aat cac tta cga tca caa tcg gtg 2544 Ala Thr Thr Thr Thr Asn Asn Asn Asn His Leu Arg Ser Gln Ser Val 835 840 845 atg tca aca tcc acg gcg acc acg atc aaa ccg agg aaa cgc aac gaa 2592 Met Ser Thr Ser Thr Ala Thr Thr Ile Lys Pro Arg Lys Arg Asn Glu 850 855 860 aat gcc aat gcc aag aag ccc aag tcc gta cat gca aag tta tcg atg 2640 Asn Ala Asn Ala Lys Lys Pro Lys Ser Val His Ala Lys Leu Ser Met 865 870 875 880 ccg ccc gaa aag atg aag tat gcg tgg tcg aat gat gtg agg aag gct 2688 Pro Pro Glu Lys Met Lys Tyr Ala Trp Ser Asn Asp Val Arg Lys Ala 885 890 895 ctc aag gat agg ttt cgg atg tcg ggg ttc aga cag aat cag ttg gag 2736 Leu Lys Asp Arg Phe Arg Met Ser Gly Phe Arg Gln Asn Gln Leu Glu 900 905 910 gct att aat gct act ttg ggt ggt aag gat gcc ttt gtg ttg atg ccg 2784 Ala Ile Asn Ala Thr Leu Gly Gly Lys Asp Ala Phe Val Leu Met Pro 915 920 925 act ggt ggt gga aag tct ctg tgc tat cag ttg ccg gct gta gtc agg 2832 Thr Gly Gly Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Val Val Arg 930 935 940 agc ggc aag acg cgt ggt atc aca gtc gtc atc tcc cct ctg cta agt 2880 Ser Gly Lys Thr Arg Gly Ile Thr Val Val Ile Ser Pro Leu Leu Ser 945 950 955 960 ctg atg ctg gat caa gtc aac cat ttg gca aac ctg atg atc caa gct 2928 Leu Met Leu Asp Gln Val Asn His Leu Ala Asn Leu Met Ile Gln Ala 965 970 975 tac gct ttc aac gga gac atg aac tca gaa atg cgc cga atg gtg ttt 2976 Tyr Ala Phe Asn Gly Asp Met Asn Ser Glu Met Arg Arg Met Val Phe 980 985 990 cag aag ctt gat gct gag cat cct gag cat gag ctc caa ctg ctc tat 3024 Gln Lys Leu Asp Ala Glu His Pro Glu His Glu Leu Gln Leu Leu Tyr 995 1000 1005 gtc acc ccg gag atg gtg agc aag aac cag aca ttc gtc aac aag atg 3072 Val Thr Pro Glu Met Val Ser Lys Asn Gln Thr Phe Val Asn Lys Met 1010 1015 1020 atg gac ctc tac cgg agg aaa aag ctg gct aga att gtt atc gac gag 3120 Met Asp Leu Tyr Arg Arg Lys Lys Leu Ala Arg Ile Val Ile Asp Glu 1025 1030 1035 1040 gct cac tgc gtc agt caa tgg ggc cat gac ttc cga ccc gat tac aaa 3168 Ala His Cys Val Ser Gln Trp Gly His Asp Phe Arg Pro Asp Tyr Lys 1045 1050 1055 gct ata gga gag ttt cgt aag agg ttt ccc gga gtt ccg gtc atg gct 3216 Ala Ile Gly Glu Phe Arg Lys Arg Phe Pro Gly Val Pro Val Met Ala 1060 1065 1070 ttg aca gcg acg gca aca cag aac gtc atc ttg gat gtc aag cat aac 3264 Leu Thr Ala Thr Ala Thr Gln Asn Val Ile Leu Asp Val Lys His Asn 1075 1080 1085 ctg gca atg gag gac tgc cag act ttc tcc cag agc ttt aat cgg ccg 3312 Leu Ala Met Glu Asp Cys Gln Thr Phe Ser Gln Ser Phe Asn Arg Pro 1090 1095 1100 aac ctc tac tat gag gtc agg atg aag gag cag aat ctg att gcc cgc 3360 Asn Leu Tyr Tyr Glu Val Arg Met Lys Glu Gln Asn Leu Ile Ala Arg 1105 1110 1115 1120 atc gca gag ttg atc aag gag aag tat gac ggc cag acg ggt atc atc 3408 Ile Ala Glu Leu Ile Lys Glu Lys Tyr Asp Gly Gln Thr Gly Ile Ile 1125 1130 1135 tac aca tta tca aga aag agt gcc gag aac atc gcc aaa aat ctc cag 3456 Tyr Thr Leu Ser Arg Lys Ser Ala Glu Asn Ile Ala Lys Asn Leu Gln 1140 1145 1150 gaa aaa cac cgc atc aaa gca aag cac tac cat gcc tcc atc acc acc 3504 Glu Lys His Arg Ile Lys Ala Lys His Tyr His Ala Ser Ile Thr Thr 1155 1160 1165 gac gaa aag atc agc gtt caa cat gaa tgg caa acc ggc cga gtc aaa 3552 Asp Glu Lys Ile Ser Val Gln His Glu Trp Gln Thr Gly Arg Val Lys 1170 1175 1180 gtc gtg gta gcc acc att gcc ttc ggc atg ggc atc gac aag cct gac 3600 Val Val Val Ala Thr Ile Ala Phe Gly Met Gly Ile Asp Lys Pro Asp 1185 1190 1195 1200 gtc cgc ttt gtt atc cac cag cac atc ccc aag tcg ctc gaa ggt tac 3648 Val Arg Phe Val Ile His Gln His Ile Pro Lys Ser Leu Glu Gly Tyr 1205 1210 1215 tat caa gaa acc ggc cgc gcc gga cgt gac ggc aag cca tcg gac tgc 3696 Tyr Gln Glu Thr Gly Arg Ala Gly Arg Asp Gly Lys Pro Ser Asp Cys 1220 1225 1230 tac ttg tac ttt gcc tat ggc gac att caa tcc cta cgt cgt atg atc 3744 Tyr Leu Tyr Phe Ala Tyr Gly Asp Ile Gln Ser Leu Arg Arg Met Ile 1235 1240 1245 gcc gac ggc gaa ggt gac tac gcg caa aag gag cgt cag cta caa atg 3792 Ala Asp Gly Glu Gly Asp Tyr Ala Gln Lys Glu Arg Gln Leu Gln Met 1250 1255 1260 ctc aac cgt gtg gtc agc tat tgc gag tcg cag cac acg tgc cgg cgc 3840 Leu Asn Arg Val Val Ser Tyr Cys Glu Ser Gln His Thr Cys Arg Arg 1265 1270 1275 1280 gaa gaa gtg ctc cgc tac ttt ggc gag gag ttt gac tac cgg aag tgt 3888 Glu Glu Val Leu Arg Tyr Phe Gly Glu Glu Phe Asp Tyr Arg Lys Cys 1285 1290 1295 aga gac gga tgc gat aac tgc cgg aac gga cgc atc tcg aag tcg acg 3936 Arg Asp Gly Cys Asp Asn Cys Arg Asn Gly Arg Ile Ser Lys Ser Thr 1300 1305 1310 gag atg agg gat ttt acg gaa atc gcc ttc gcc gcg atc gag gtg gtg 3984 Glu Met Arg Asp Phe Thr Glu Ile Ala Phe Ala Ala Ile Glu Val Val 1315 1320 1325 aag agc cag cag ccc atc acg ctg ggc aag ctg tgc gac atc ctg atg 4032 Lys Ser Gln Gln Pro Ile Thr Leu Gly Lys Leu Cys Asp Ile Leu Met 1330 1335 1340 ggc aag aga aag aac gag cac ggt ggc gtg tgt cac ttt ggt atc gcc 4080 Gly Lys Arg Lys Asn Glu His Gly Gly Val Cys His Phe Gly Ile Ala 1345 1350 1355 1360 aag ggg agc acg cag agg gag ctg cag agg atc gtg ctg cag ctg aat 4128 Lys Gly Ser Thr Gln Arg Glu Leu Gln Arg Ile Val Leu Gln Leu Asn 1365 1370 1375 ttc cac aag gcg ctg ggc gag gac aat atc atg aat ggg gcg ggg atg 4176 Phe His Lys Ala Leu Gly Glu Asp Asn Ile Met Asn Gly Ala Gly Met 1380 1385 1390 cct att acc tac tat att acc ggc cct gaa gct ggt gct tac ctc tac 4224 Pro Ile Thr Tyr Tyr Ile Thr Gly Pro Glu Ala Gly Ala Tyr Leu Tyr 1395 1400 1405 aat ggc aag cgg ttg atg ctg cca gtt ccc tca aac aag tcc gtc gaa 4272 Asn Gly Lys Arg Leu Met Leu Pro Val Pro Ser Asn Lys Ser Val Glu 1410 1415 1420 ccc ccg tct cgg tct aag cag cgg agc cgt cga gtc gat gag gat atg 4320 Pro Pro Ser Arg Ser Lys Gln Arg Ser Arg Arg Val Asp Glu Asp Met 1425 1430 1435 1440 gat gag caa gaa ctt tcc acc ctg caa cga ccg cca aca tca aca aat 4368 Asp Glu Gln Glu Leu Ser Thr Leu Gln Arg Pro Pro Thr Ser Thr Asn 1445 1450 1455 gtc tct tca ccc gtt cga gcc acc aag aaa cga agt tcc aaa aag gct 4416 Val Ser Ser Pro Val Arg Ala Thr Lys Lys Arg Ser Ser Lys Lys Ala 1460 1465 1470 tta ccg acc ctc atc gcc gac tac gaa gag ccc agc tcc gac ggt cct 4464 Leu Pro Thr Leu Ile Ala Asp Tyr Glu Glu Pro Ser Ser Asp Gly Pro 1475 1480 1485 cac ggt cct ctc cac gcc aac ggc tat gag cgc gat aac ttt gtc gta 4512 His Gly Pro Leu His Ala Asn Gly Tyr Glu Arg Asp Asn Phe Val Val 1490 1495 1500 tcc gat aat gtt gaa ccc gaa gag gaa gaa gat gcc ttc gaa cct gtc 4560 Ser Asp Asn Val Glu Pro Glu Glu Glu Glu Asp Ala Phe Glu Pro Val 1505 1510 1515 1520 cgc ccc tcg cgg cgc ggc cca tct tct cgc gct acc cgc cct caa cac 4608 Arg Pro Ser Arg Arg Gly Pro Ser Ser Arg Ala Thr Arg Pro Gln His 1525 1530 1535 cgc cag acc acc ctt tat gac acc ctc tcc cac acc caa caa tcc caa 4656 Arg Gln Thr Thr Leu Tyr Asp Thr Leu Ser His Thr Gln Gln Ser Gln 1540 1545 1550 acc gtc tcc caa cac ctc gcc act ttg ggt ccg ccc atc gac gcc cgc 4704 Thr Val Ser Gln His Leu Ala Thr Leu Gly Pro Pro Ile Asp Ala Arg 1555 1560 1565 acc atg cat aac ccc cgc tac gcc cag ctt gac gag gtc cac cag gat 4752 Thr Met His Asn Pro Arg Tyr Ala Gln Leu Asp Glu Val His Gln Asp 1570 1575 1580 att gtc gat gcc ttt gtt gaa gaa gtc aag gtc ttc gag gag gac ttt 4800 Ile Val Asp Ala Phe Val Glu Glu Val Lys Val Phe Glu Glu Asp Phe 1585 1590 1595 1600 cgc aac agg aac cac atg cgc aaa ccc atc ttt acc gag acg cag tac 4848 Arg Asn Arg Asn His Met Arg Lys Pro Ile Phe Thr Glu Thr Gln Tyr 1605 1610 1615 cgt gag atg gca atc cgg tgg acg cgg tcg tta gac gcg atg cgc gcg 4896 Arg Glu Met Ala Ile Arg Trp Thr Arg Ser Leu Asp Ala Met Arg Ala 1620 1625 1630 atc ccg gat atc aac cag gat aaa gta gat cgg tat ggt gcc aaa ttc 4944 Ile Pro Asp Ile Asn Gln Asp Lys Val Asp Arg Tyr Gly Ala Lys Phe 1635 1640 1645 atc cca ctt gtg gag cgg ttc tgg ggg aat tat cag gag atg atg ggg 4992 Ile Pro Leu Val Glu Arg Phe Trp Gly Asn Tyr Gln Glu Met Met Gly 1650 1655 1660 gga ggg tat gat aat cct gct gtg gct ggc gat gag gat gat gat gag 5040 Gly Gly Tyr Asp Asn Pro Ala Val Ala Gly Asp Glu Asp Asp Asp Glu 1665 1670 1675 1680 ggc ccc agg agg aca gga aat gga aaa ggg ggg aat aag aag gga gga 5088 Gly Pro Arg Arg Thr Gly Asn Gly Lys Gly Gly Asn Lys Lys Gly Gly 1685 1690 1695 gga gga gga gga gga aat gaa gta gtg gat ttg att agt agt gat gag 5136 Gly Gly Gly Gly Gly Asn Glu Val Val Asp Leu Ile Ser Ser Asp Glu 1700 1705 1710 gat gaa cct ccg gct cgt gca cca tcg cgg aat gcg ggg cga gga aag 5184 Asp Glu Pro Pro Ala Arg Ala Pro Ser Arg Asn Ala Gly Arg Gly Lys 1715 1720 1725 gca cag tcg aca cgt ggg gga caa atc caa gat aaa ggc cga gca gtc 5232 Ala Gln Ser Thr Arg Gly Gly Gln Ile Gln Asp Lys Gly Arg Ala Val 1730 1735 1740 aac cgc cgc gga gaa ccc atc gcc gaa gaa gac gaa gaa gac tac ggg 5280 Asn Arg Arg Gly Glu Pro Ile Ala Glu Glu Asp Glu Glu Asp Tyr Gly 1745 1750 1755 1760 cta agc gac ccc gat atc gac gcc atc gat cca gac gcc atc acc gcc 5328 Leu Ser Asp Pro Asp Ile Asp Ala Ile Asp Pro Asp Ala Ile Thr Ala 1765 1770 1775 tcc gac aac tcc gac gaa gaa gat gat gat gat gat gac gaa gac ctc 5376 Ser Asp Asn Ser Asp Glu Glu Asp Asp Asp Asp Asp Asp Glu Asp Leu 1780 1785 1790 gaa tcc tcc cgc tac ttc tcc ggc tca aca ggc ccg ccc gtc tcc aaa 5424 Glu Ser Ser Arg Tyr Phe Ser Gly Ser Thr Gly Pro Pro Val Ser Lys 1795 1800 1805 gcc gtg cag gat gct cga ctc cgt gaa caa ctt tcc atg tac gcc tcc 5472 Ala Val Gln Asp Ala Arg Leu Arg Glu Gln Leu Ser Met Tyr Ala Ser 1810 1815 1820 ggc ggc agc tct tcg aaa ggt agc tac ggc tca ggg cgc gca tca gga 5520 Gly Gly Ser Ser Ser Lys Gly Ser Tyr Gly Ser Gly Arg Ala Ser Gly 1825 1830 1835 1840 gga tct tcg tcg aga gcg tcg gga tca gga tgg aga ggt gga gga gca 5568 Gly Ser Ser Ser Arg Ala Ser Gly Ser Gly Trp Arg Gly Gly Gly Ala 1845 1850 1855 ggt ggg aag aaa tac tac agg aag aag agg gct ggt tct tcg gct gct 5616 Gly Gly Lys Lys Tyr Tyr Arg Lys Lys Arg Ala Gly Ser Ser Ala Ala 1860 1865 1870 ggt ggt ggt ggt gca gga gga ggg gga gtt aca aaa cgg aag gcg agt 5664 Gly Gly Gly Gly Ala Gly Gly Gly Gly Val Thr Lys Arg Lys Ala Ser 1875 1880 1885 ggg agt ggc gcg aag acg gcg agg aag agg ggt gca tct act gcg ccg 5712 Gly Ser Gly Ala Lys Thr Ala Arg Lys Arg Gly Ala Ser Thr Ala Pro 1890 1895 1900 aag aca acg acg aga ggg gga gga tct gga gct ggg tct aga gga ggc 5760 Lys Thr Thr Thr Arg Gly Gly Gly Ser Gly Ala Gly Ser Arg Gly Gly 1905 1910 1915 1920 ggt gct ggt ggt gct ggt ggt gct ggt gct ggt gct ggt gct gga gga 5808 Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly 1925 1930 1935 ggg aaa agg ggt ggt gga ggt gga gga gga atg gga ggg ata agt gtt 5856 Gly Lys Arg Gly Gly Gly Gly Gly Gly Gly Met Gly Gly Ile Ser Val 1940 1945 1950 atg cct cat tag 5868 Met Pro His 1955 3 1955 PRT Neurospora crassa 3 Met Ala Lys Leu Ser Val Lys Asn Asn Leu Pro Arg Pro His Leu Val 1 5 10 15 Ser Leu Ser Ser Ser Thr Thr Gly Ser Gly Ser Gly Ser Ala Ser Arg 20 25 30 Ser Ala Ser Ala Lys His Gly Ser Ala Gly Ser Ser Thr Phe Asp His 35 40 45 Glu Gln His Gln Gln His Gln Gln Gln Gln Gln Gln Lys Arg Gln Arg 50 55 60 Ser Gln Ser Glu Ala Arg Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 65 70 75 80 Gln Gln Gln Gln Gln Gln Gln Gln Ala Gln His His Ala His Ser Thr 85 90 95 Tyr Ala Gln Arg Pro Gln Pro Thr Pro Gln Gln Arg Pro Pro Gln Asn 100 105 110 Leu Leu Thr Pro Ala Ser Thr Thr Gly Ala Ser Val Gly Pro Leu Gln 115 120 125 Arg Ala Tyr Ser Val Ser Leu Ala Ala Arg Gln Ser Pro Ser Thr Asn 130 135 140 Leu Val Arg Pro Lys Thr Asp Ser Pro Ala Pro His Thr Leu His Leu 145 150 155 160 Lys Asn Lys Lys Asn Leu Arg His Pro Ala Pro Thr Pro Asp Ser Pro 165 170 175 Ile Val Asp Asp Asp Ile Phe Ser Asp Ala Val Asp Leu Thr Glu Glu 180 185 190 Leu Asp His Asp His Asp Leu Asn Gly Lys Asp Lys Asp Asn Thr Asp 195 200 205 Asn Asp Asn Thr Val Ala Ser Ser Ser Leu Ile Gly Phe Gly Asp Asp 210 215 220 Lys Leu Leu Trp Arg Glu Asp Phe Ala Glu Arg Ala Glu Pro Glu His 225 230 235 240 Glu Arg Gly Gly Ser Arg Pro Arg Gln Val Lys Lys Arg Lys Ile Ser 245 250 255 Asn Asp Tyr Ile Met Lys Asp Glu Asp Val Ser Leu Phe Asp Asp Asp 260 265 270 Gly Glu Glu Asp Glu Phe Met Asp Ile Asn Glu Leu Val Gln Gly Asp 275 280 285 Arg Glu Ser Thr Pro Lys Pro Lys Ala Thr Ser Arg Ser Val Ser Thr 290 295 300 Arg Leu Pro Pro Thr Val Ser Leu Gln Arg Gly Arg Ser Pro Lys Arg 305 310 315 320 Lys Glu Ala Ser Val Glu Lys Arg Thr Thr Glu Asn Gln Gln Gln Ala 325 330 335 Asp Arg Glu Asp Glu Pro Ser Phe Met Ser Ser Pro Asp Val Asp Asn 340 345 350 Ser Arg Lys Arg Lys Ser Ser Gly Ser Pro Thr Gly Leu Thr Thr Pro 355 360 365 Arg Pro Gln Gln Lys Gln Thr Glu Glu Val Pro Gly Thr Thr Thr Ala 370 375 380 Lys Lys Pro Arg Arg Ser Glu Val Met Asp Ser Glu Asp Glu Ala Phe 385 390 395 400 Thr Pro Leu Ser Ala Gly Ser Leu Pro Gly Ser Ala Glu Phe Phe Arg 405 410 415 Ser Gly Gly Thr Thr Thr Arg Glu Leu Gly Leu Asp Glu Asp Thr Val 420 425 430 Met Asp Thr Pro Ser Arg Pro Pro Val Glu Ser Thr Leu Pro Thr Leu 435 440 445 Glu Ser Val Glu Ser Arg Pro Pro Pro Leu Pro Pro Met Asp Leu Pro 450 455 460 Ser Gln Arg Lys Pro Leu Glu Pro Leu Asn Thr Pro Arg Asn Gln Leu 465 470 475 480 Leu Glu Ser Val Glu Arg Pro Thr Gln Gln Pro Ser Val Gly Pro Ser 485 490 495 Phe Ala Gln Ser Ser Thr Leu Ala Glu Ser Ser Leu Pro Pro Ser Met 500 505 510 Pro Pro Pro Ser Glu Asp Pro Leu Asn Thr Arg Glu Asn Ser Asn Leu 515 520 525 Glu Glu Phe Asp Tyr Lys Leu Tyr Lys Pro Leu Leu Asp Leu Phe Val 530 535 540 Asn Ala Pro Ala Ile Leu Glu Arg Glu Leu Ser Ala Val Asn Asp Glu 545 550 555 560 Leu Gln Glu Asn Met Ile Lys Leu Arg Asp Cys Leu Arg Leu Pro Arg 565 570 575 Glu Glu Arg Asp Arg Ala Arg Glu Glu Val Lys Lys Glu Lys Glu Met 580 585 590 Leu Lys Arg Arg Asp Ile Ala Leu Arg Ala Leu Gln Asp Glu His Lys 595 600 605 Leu Tyr Val Lys Lys Arg Lys Glu His Asn Leu Ile Asn Glu Glu Ile 610 615 620 Val Arg Ala Tyr Ala Glu Glu Asp Asp Glu Tyr Glu Asp Gln Leu Met 625 630 635 640 Ala Gln Leu Asp Lys Leu Asp Asp Glu Val Glu Ala Ile Val Lys Ser 645 650 655 Leu Thr Arg Leu Ile Val Ala Ala Gly Ile Thr Glu Lys Ser Phe Asp 660 665 670 Leu Lys Lys Glu Glu Glu Glu Glu Glu Glu Lys Pro Ile Ile Ile Ala 675 680 685 Thr Pro Thr Pro Ser Thr Arg Thr Glu Ala Pro Val Leu Pro Thr Thr 690 695 700 Glu Tyr His Asn Ser Gln Gln Val Ile Leu Gln Thr Gln His Pro Ala 705 710 715 720 Ala Gln Gln Val Ser His Arg Val Pro Pro Pro Pro Thr Pro Ser Phe 725 730 735 Gln Thr Ala Arg Gln Thr Pro Val Ser Tyr Gln Ser Arg Pro Thr Asn 740 745 750 Asn Ser Phe Pro Asp Ile Ser Ala Glu Glu Ala Met Met Phe Asp Lys 755 760 765 Glu Asp Pro Phe Met Glu Gln Gln His Ala Pro Ala Ser Ala Pro Phe 770 775 780 Gln Ala Thr Leu Pro Gln Arg Asn Ser Pro Phe Lys Thr Ala Pro Phe 785 790 795 800 Lys Pro Val His Gly His Asp Tyr Phe Asp Asp Glu Asp Asp Asp Ala 805 810 815 Asp Leu Leu Ala Ala Val Asp Ser Ala Glu Thr Tyr Thr Ser Thr Ala 820 825 830 Ala Thr Thr Thr Thr Asn Asn Asn Asn His Leu Arg Ser Gln Ser Val 835 840 845 Met Ser Thr Ser Thr Ala Thr Thr Ile Lys Pro Arg Lys Arg Asn Glu 850 855 860 Asn Ala Asn Ala Lys Lys Pro Lys Ser Val His Ala Lys Leu Ser Met 865 870 875 880 Pro Pro Glu Lys Met Lys Tyr Ala Trp Ser Asn Asp Val Arg Lys Ala 885 890 895 Leu Lys Asp Arg Phe Arg Met Ser Gly Phe Arg Gln Asn Gln Leu Glu 900 905 910 Ala Ile Asn Ala Thr Leu Gly Gly Lys Asp Ala Phe Val Leu Met Pro 915 920 925 Thr Gly Gly Gly Lys Ser Leu Cys Tyr Gln Leu Pro Ala Val Val Arg 930 935 940 Ser Gly Lys Thr Arg Gly Ile Thr Val Val Ile Ser Pro Leu Leu Ser 945 950 955 960 Leu Met Leu Asp Gln Val Asn His Leu Ala Asn Leu Met Ile Gln Ala 965 970 975 Tyr Ala Phe Asn Gly Asp Met Asn Ser Glu Met Arg Arg Met Val Phe 980 985 990 Gln Lys Leu Asp Ala Glu His Pro Glu His Glu Leu Gln Leu Leu Tyr 995 1000 1005 Val Thr Pro Glu Met Val Ser Lys Asn Gln Thr Phe Val Asn Lys Met 1010 1015 1020 Met Asp Leu Tyr Arg Arg Lys Lys Leu Ala Arg Ile Val Ile Asp Glu 1025 1030 1035 1040 Ala His Cys Val Ser Gln Trp Gly His Asp Phe Arg Pro Asp Tyr Lys 1045 1050 1055 Ala Ile Gly Glu Phe Arg Lys Arg Phe Pro Gly Val Pro Val Met Ala 1060 1065 1070 Leu Thr Ala Thr Ala Thr Gln Asn Val Ile Leu Asp Val Lys His Asn 1075 1080 1085 Leu Ala Met Glu Asp Cys Gln Thr Phe Ser Gln Ser Phe Asn Arg Pro 1090 1095 1100 Asn Leu Tyr Tyr Glu Val Arg Met Lys Glu Gln Asn Leu Ile Ala Arg 1105 1110 1115 1120 Ile Ala Glu Leu Ile Lys Glu Lys Tyr Asp Gly Gln Thr Gly Ile Ile 1125 1130 1135 Tyr Thr Leu Ser Arg Lys Ser Ala Glu Asn Ile Ala Lys Asn Leu Gln 1140 1145 1150 Glu Lys His Arg Ile Lys Ala Lys His Tyr His Ala Ser Ile Thr Thr 1155 1160 1165 Asp Glu Lys Ile Ser Val Gln His Glu Trp Gln Thr Gly Arg Val Lys 1170 1175 1180 Val Val Val Ala Thr Ile Ala Phe Gly Met Gly Ile Asp Lys Pro Asp 1185 1190 1195 1200 Val Arg Phe Val Ile His Gln His Ile Pro Lys Ser Leu Glu Gly Tyr 1205 1210 1215 Tyr Gln Glu Thr Gly Arg Ala Gly Arg Asp Gly Lys Pro Ser Asp Cys 1220 1225 1230 Tyr Leu Tyr Phe Ala Tyr Gly Asp Ile Gln Ser Leu Arg Arg Met Ile 1235 1240 1245 Ala Asp Gly Glu Gly Asp Tyr Ala Gln Lys Glu Arg Gln Leu Gln Met 1250 1255 1260 Leu Asn Arg Val Val Ser Tyr Cys Glu Ser Gln His Thr Cys Arg Arg 1265 1270 1275 1280 Glu Glu Val Leu Arg Tyr Phe Gly Glu Glu Phe Asp Tyr Arg Lys Cys 1285 1290 1295 Arg Asp Gly Cys Asp Asn Cys Arg Asn Gly Arg Ile Ser Lys Ser Thr 1300 1305 1310 Glu Met Arg Asp Phe Thr Glu Ile Ala Phe Ala Ala Ile Glu Val Val 1315 1320 1325 Lys Ser Gln Gln Pro Ile Thr Leu Gly Lys Leu Cys Asp Ile Leu Met 1330 1335 1340 Gly Lys Arg Lys Asn Glu His Gly Gly Val Cys His Phe Gly Ile Ala 1345 1350 1355 1360 Lys Gly Ser Thr Gln Arg Glu Leu Gln Arg Ile Val Leu Gln Leu Asn 1365 1370 1375 Phe His Lys Ala Leu Gly Glu Asp Asn Ile Met Asn Gly Ala Gly Met 1380 1385 1390 Pro Ile Thr Tyr Tyr Ile Thr Gly Pro Glu Ala Gly Ala Tyr Leu Tyr 1395 1400 1405 Asn Gly Lys Arg Leu Met Leu Pro Val Pro Ser Asn Lys Ser Val Glu 1410 1415 1420 Pro Pro Ser Arg Ser Lys Gln Arg Ser Arg Arg Val Asp Glu Asp Met 1425 1430 1435 1440 Asp Glu Gln Glu Leu Ser Thr Leu Gln Arg Pro Pro Thr Ser Thr Asn 1445 1450 1455 Val Ser Ser Pro Val Arg Ala Thr Lys Lys Arg Ser Ser Lys Lys Ala 1460 1465 1470 Leu Pro Thr Leu Ile Ala Asp Tyr Glu Glu Pro Ser Ser Asp Gly Pro 1475 1480 1485 His Gly Pro Leu His Ala Asn Gly Tyr Glu Arg Asp Asn Phe Val Val 1490 1495 1500 Ser Asp Asn Val Glu Pro Glu Glu Glu Glu Asp Ala Phe Glu Pro Val 1505 1510 1515 1520 Arg Pro Ser Arg Arg Gly Pro Ser Ser Arg Ala Thr Arg Pro Gln His 1525 1530 1535 Arg Gln Thr Thr Leu Tyr Asp Thr Leu Ser His Thr Gln Gln Ser Gln 1540 1545 1550 Thr Val Ser Gln His Leu Ala Thr Leu Gly Pro Pro Ile Asp Ala Arg 1555 1560 1565 Thr Met His Asn Pro Arg Tyr Ala Gln Leu Asp Glu Val His Gln Asp 1570 1575 1580 Ile Val Asp Ala Phe Val Glu Glu Val Lys Val Phe Glu Glu Asp Phe 1585 1590 1595 1600 Arg Asn Arg Asn His Met Arg Lys Pro Ile Phe Thr Glu Thr Gln Tyr 1605 1610 1615 Arg Glu Met Ala Ile Arg Trp Thr Arg Ser Leu Asp Ala Met Arg Ala 1620 1625 1630 Ile Pro Asp Ile Asn Gln Asp Lys Val Asp Arg Tyr Gly Ala Lys Phe 1635 1640 1645 Ile Pro Leu Val Glu Arg Phe Trp Gly Asn Tyr Gln Glu Met Met Gly 1650 1655 1660 Gly Gly Tyr Asp Asn Pro Ala Val Ala Gly Asp Glu Asp Asp Asp Glu 1665 1670 1675 1680 Gly Pro Arg Arg Thr Gly Asn Gly Lys Gly Gly Asn Lys Lys Gly Gly 1685 1690 1695 Gly Gly Gly Gly Gly Asn Glu Val Val Asp Leu Ile Ser Ser Asp Glu 1700 1705 1710 Asp Glu Pro Pro Ala Arg Ala Pro Ser Arg Asn Ala Gly Arg Gly Lys 1715 1720 1725 Ala Gln Ser Thr Arg Gly Gly Gln Ile Gln Asp Lys Gly Arg Ala Val 1730 1735 1740 Asn Arg Arg Gly Glu Pro Ile Ala Glu Glu Asp Glu Glu Asp Tyr Gly 1745 1750 1755 1760 Leu Ser Asp Pro Asp Ile Asp Ala Ile Asp Pro Asp Ala Ile Thr Ala 1765 1770 1775 Ser Asp Asn Ser Asp Glu Glu Asp Asp Asp Asp Asp Asp Glu Asp Leu 1780 1785 1790 Glu Ser Ser Arg Tyr Phe Ser Gly Ser Thr Gly Pro Pro Val Ser Lys 1795 1800 1805 Ala Val Gln Asp Ala Arg Leu Arg Glu Gln Leu Ser Met Tyr Ala Ser 1810 1815 1820 Gly Gly Ser Ser Ser Lys Gly Ser Tyr Gly Ser Gly Arg Ala Ser Gly 1825 1830 1835 1840 Gly Ser Ser Ser Arg Ala Ser Gly Ser Gly Trp Arg Gly Gly Gly Ala 1845 1850 1855 Gly Gly Lys Lys Tyr Tyr Arg Lys Lys Arg Ala Gly Ser Ser Ala Ala 1860 1865 1870 Gly Gly Gly Gly Ala Gly Gly Gly Gly Val Thr Lys Arg Lys Ala Ser 1875 1880 1885 Gly Ser Gly Ala Lys Thr Ala Arg Lys Arg Gly Ala Ser Thr Ala Pro 1890 1895 1900 Lys Thr Thr Thr Arg Gly Gly Gly Ser Gly Ala Gly Ser Arg Gly Gly 1905 1910 1915 1920 Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly 1925 1930 1935 Gly Lys Arg Gly Gly Gly Gly Gly Gly Gly Met Gly Gly Ile Ser Val 1940 1945 1950 Met Pro His 1955 

1. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and in comprising a recQ helicase domain, wherein the domain is at least 30% homologous with the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 2. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 1, wherein the domain is at least 40% homologous with the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 3. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 2, wherein the domain is at least 60% homologous with the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 4. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 3, wherein the recQ helicase domain has the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 5. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 4, wherein said nucleotide sequence encodes for a protein having the amino acid sequence of SEQ ID No. 1, or functional portions thereof.
 6. An isolated nucleic acid molecule encoding for a protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 5, wherein said nucleotide sequence is the sequence of SEQ ID No. 1 or its complementary sequence.
 7. Expression vector comprising, under the control of a promoter that directs the expression of genes in bacteria, the isolated nucleic acid molecule according to any one of claims 1-6.
 8. Expression vector comprising, under the control of a promoter that directs the expression of genes in plants or in specific plant organs, the isolated nucleic acid molecule according to any one of claims 1-6, both in a sense and anti-sense orientation.
 9. Expression vector comprising, under the control of a promoter that directs the expression of genes in fungi, the isolated nucleic acid molecule according to any one of claims 1-6 both in a sense and anti-sense orientation.
 10. Expression vector comprising, under the control of a promoter that is expressed in animals, the isolated nucleic acid molecule according to any one of claims 1-6 both in a sense and anti-sense orientation.
 11. Prokaryotic organism transformed by using the expression vector active in bacteria according to claim
 7. 12. Plants or a specific plant organ transformed by using the expression vector active in plants according to claim
 8. 13. Plant mutated at the nucleotide sequence according to any one of claims 1-6 having a reduced or inhibited silencing activity.
 14. Fungus transformed by using the expression vector active in fungi according to claim
 9. 15. Non-human animal transformed by using the expression vector active in animals according to claim
 10. 16. Non-human animal mutated at the nucleotide sequence according to any one of claims 1-6 having a reduced or inhibited silencing activity.
 17. Protein characterized in having a silencing activity and comprising a recQ helicase domain wherein the domain is at least 30% homologous to the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 18. Protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 17 wherein the domain is at least 40% homologous to the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 19. Protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 18 wherein the domain is at least 60% homologous to the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 20. Protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 19 wherein the domain is the amino acid sequence from aa. 897 to aa. 1330 of SEQ ID No.1.
 21. Protein characterized in having a silencing activity and comprising a recQ helicase domain according to claim 21 comprising the amino acid sequence of SEQ ID. No.1 or functional portions thereof.
 22. Use of the nucleotide sequence according to any one of claims 1-6 to modulate the gene silencing in plants, animals and fungi.
 23. Use of the nucleotide sequence according to any one of claims 1-6 to potentiate the antiviral-response in a plant. 